Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli

A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp. aizawai IPL7 was cloned from a 78-kb plasmid. The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions. The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues. Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions. It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli. The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene. Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein.     

Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon

The complete nucleotide sequence of the Corynebacterium glutamicum hom-thrB operon has been determined and the structural genes and promoter region mapped. A polypeptide of Mr 46,136 is encoded by hom and a polypeptide of Mr 32,618 is encoded by thrB. Both predicted protein sequences show amino acid sequence homology to their counterparts in Escherichia coli and Bacillus subtilis. The promoter region has been mapped by S1-nuclease and deletion analysis. Located between -88, RNA start site and -219 (smallest deletion clone with complete activity) are sequence elements similar to those found in E. coli and B. subtilis promoters. Although there are no obvious attenuator-like structures in the 5'-untranslated region, there is a dyad-symmetry element, which may act as an operator.     

Nucleotide sequence of the extracellular glucoamylase gene STA1 in the yeast Saccharomyces diastaticus.

The complete nucleotide sequence of the extracellular glucoamylase gene STA1 from the yeast Saccharomyces diastaticus has been determined. A single open reading frame codes for a 778-amino-acid protein which contains 13 potential N-glycosylation sites. In the 5'- and 3'-flanking regions of the gene, there are striking sequence homologies to the corresponding regions of ADH1 for alcohol dehydrogenase and MAT alpha 2 for mating type control in the yeast Saccharomyces cerevisiae. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. The presumptive signal sequence showed a significant homology to that of Bacillus subtilis alpha-amylase precursor. The next segment, of ca. 320 amino acids, contains a threonine-rich tract in which direct repeat sequences of 35 amino acids exist, and is bordered by a pair of basic amino acid residues (Lys-Lys) which may be a proteolytic processing signal. The carboxy-terminal half of the precursor is a presumptive glucoamylase which contains several peptide segments showing a high degree of homology with alpha-amylases from widely diverse organisms including a procaryote (B. subtilis) and eucaryotes (Aspergillus oryzae and mouse). Analysis of both the nucleotide sequence of the STA1 gene and the amino acid composition of the purified glucoamylase suggested that the putative precursor is processed to yield subunits H and Y of mature enzyme by both trypsin-like and chymotrypsin-like cleavages.     

Sequencing and heterologous expression of the gene encoding penicillin V amidase from Bacillus sphaericus

The Bacillus sphaericus gene encoding penicillin V amidase, which catalyzes the hydrolysis of penicillin V, has been characterized. The entire nucleotide sequence of the coding region, as well as 5'- and 3'-flanking regions, was determined using an improved sequencing strategy. The deduced amino acid sequence suggests a protein consisting of 338 residues with an Mr of 37,500. The ATG initiator codon is preceded by a putative ribosome-binding site, typical for genes of Gram-positive origin. High expression of the gene was obtained in Escherichia coli using an inducible promoter, showing that the gene product is stable in this heterologous host.     

枯草芽孢杆菌渗透压调节基因proB的克隆和表达

用PCR扩增的方法从耐盐的枯草杆菌中克隆出一个13kb长的DNA片段,经功能检测,证明正向插入片段与大肠杆菌的脯氨酸营养缺陷特性(proB-)能够营养互补。含有该重组质粒的大肠杆菌DH5α在基本培养基上的耐盐能力从2%提高至4%。通过引物步行法测定了该插入片段的核苷酸序列。利用DNAsis软件进行序列分析发现,该片段第122～1235bp核苷酸编码一个由370个氨基酸组成的蛋白质分子,其上游存在非典型的-10区,典型的-35区和核糖体结合位点,起始密码子处有最佳翻译起始效率的侧翼核苷酸序列。将其与Genebank中的已知基因的序列和编码的氨基酸序列进行同源性比较,结果表明该片段与枯草杆菌168的核苷酸序列、氨基酸序列的同源性分别为81%和90%。证明该基因确实是一个proB基因。通过与三十个不同种属微芽生物proB基因的氨基酸序列比较,发现该蛋白存在有可能与形成酶的活性中心和三维结构有密切关系的几个绝对保守的区域。     

Cloning and sequencing of Serratia protease gene.

The gene encoding an extracellular metalloproteinase from Serratia sp. E-15 has been cloned, and its complete nucleotide sequence determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature protein of the Serratia protease consists of 470 amino acids with a molecular weight of 50,632. The G+C content of the coding region for the mature protein is 58%; this high G+C content is due to a marked preference for G+C bases at the third position of the codons. The gene codes for a short pro-peptide preceding the mature protein. The Serratia protease gene was expressed in Escherichia coli and Serratia marcescens; the former produced the Serratia protease in the cells and the latter in the culture medium. Three zinc ligands and an active site of the Serratia protease were predicted by comparing the structure of the enzyme with those of thermolysin and Bacillus subtilis neutral protease.     

Nucleotide sequence of a cellulase gene of Bacillus subtilis

The nucleotide sequence of an endolytic cellulase gene of Bacillus subtilis was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme. The mature protein appeared to be extended by a signal sequence of 36 amino acids. The putative AUG initiation codon was preceded by a sigma 43-type promoter of B. subtilis and an AAGGAGG sequence, typical of procaryotic ribosomal binding sites. Partial homology of amino acid sequences was found between B. subtilis cellulase and an alkalophilic Bacillus cellulase.     

Cloning and expression of subtilisin amylosacchariticus gene

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.     

Cloning and sequencing of a gene from Bacillus amyloliquefaciens that complements mutations of the sporulation gene spoIID in Bacillus subtilis

A segment of DNA from Bacillus amyloliquefaciens, which complemented a mutant sporulation gene, spoIID68, in Bacillus subtilis, was cloned into a derivative of the temperate bacteriophage phi 105. The segment of DNA included an entire structural gene and complemented the mutation spoIID298, in addition to spoIID68, in B. subtilis. The nucleotide sequence of the gene from B. amyloliquefaciens was determined and compared with that of the B. subtilis gene; 74% homology was found in the coding region. Amino acid primary sequences derived from the nucleotide sequences of the two genes were also compared. The gene from B. amyloliquefaciens coded for a protein of 344 amino acid residues, one more than the protein coded by the corresponding gene from B. subtilis. Comparison of the primary amino acid sequences of the two genes showed that 78% of the residues were completely conserved and 8% were semi-conserved. Variation, however, was not random, i.e. some segments were much more highly conserved than others. Both proteins had a hydrophobic region at the N-terminus.     

Nucleotide sequence of the thymidylate synthetase gene (thyP3) from the Bacillus subtilis phage phi 3T

The thyP3 gene, encoding thymidylate synthetase, from the Bacillus subtilis phage phi 3T has been cloned and the nucleotide sequence determined. The derived amino acid sequence indicates a subunit Mr of 32 748. The primary amino acid sequence is compared with the sequences of the analogous proteins specified by Escherichia coli (thyA), Lactobacillus casei, (thyA) and phage T4 (td). Extensive conservation exists in all four sequences implying a shared tertiary structure.     

Gene cloning, nucleotide sequence, and expression of a cephalosporin-C deacetylase from Bacillus subtilis.

The gene encoding a cephalosporin-C deacetylase (CAH) from Bacillus subtilis SHS 0133 was cloned and sequenced. The nucleotide sequence contained an open reading frame encoding a polypeptide consisting of 318 amino acids, the molecular weight of which was in good agreement with the value obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence contained the common sequence Gly-X-Ser-X-Gly found in many esterases, lipases, and serine proteases. This indicates that CAH is a serine enzyme. A possible promoter sequence which is very similar to the consensus sequences of -35 and -10 regions recognized by B. subtilis RNA polymerase utilizing sigma factor H was found in the 5'-flanking region of the CAH structural gene. Two repeated A+T-rich blocks consisting of 24 bp were also found in the upstream region of the initiation codon. We constructed a series of expression plasmids by inserting the CAH gene into Escherichia coli ATG vectors. The degree of CAH gene expression depended on promoters and vector plasmids, which have different replication origins. The expressed CAH protein was an active form in the soluble fraction obtained after cell disruption. The highest expression level was accomplished with an expression plasmid, pCAH400, which has the trp promoter and the replication origin derived from pAT153. In the fermentation using a 30-liter jar fermentor, the transformant E. coli JM103(pCAH400) produced 440 U of CAH per ml of culture during a 24-h incubation. This value corresponded to 2.1 g of CAH protein in 1 liter of culture broth.     

Thermostable alanine racemase from Bacillus stearothermophilus: DNA and protein sequence determination and secondary structure prediction

The nucleotide sequence of the alanine racemase (EC 5.1.1.1) gene from a thermophile, Bacillus stearothermophilus, was determined by the dideoxy chain termination method with universal and synthetic site-specific primers. The amino acid sequence of the enzyme predicted from the nucleotide sequence was confirmed by peptide sequence information derived from the N-terminal amino acid residues and several tryptic fragments. The alanine racemase gene consists of 1158 base pairs encoding a protein of 386 amino acid residues; the molecular weight of the apoenzyme is estimated as 43,341. The racemase gene of B. stearothermophilus has a closely similar size (1158 vs 1167 base pairs) to that of the gene of a mesophile, B. subtilis, but shows a higher preference for codons ending in G or C. A comparison of the amino acid sequence with those of Bacillus subtilis and Salmonella typhimurium dadB and alr enzymes revealed overall sequence homologies of 31-54%, including an identical octapeptide bearing the pyridoxal 5'-phosphate binding site. Although the residues common in the four racemases are not continuously arrayed, these constitute distinct domains and their hydropathy profiles are very similar. The secondary structure of B. stearothermophilus alanine racemase was predicted from the results obtained by theoretical analysis and circular dichroism measurement.     

Alpha-amylase genes (amyR2 and amyE+) from an alpha-amylase-hyperproducing Bacillus subtilis strain: molecular cloning and nucleotide sequences.

amyR2, amyE+, and aroI+ alleles from an alpha-amylase-hyperproducing strain, Bacillus subtilis NA64, were cloned in temperate B. subtilis phage p11, and the amyR2 and amyE+ genes were then recloned in plasmid pUB110, which was designated pTUB4. The order of the restriction sites, ClaI-EcoRI-PstI-SalI-SmaI, found in the DNA fragment carrying amyR2 and amyE+ from the phage genome was also found in the 2.3-kilobase insert of pTUB4. Approximately 2,600 base pairs of the DNA nucleotide sequence of the amyR2 and amyE+ gene region in pTUB4 were determined. Starting from an ATG initiator codon, an open reading frame was composed of a total 1,776 base pairs (592 amino acids). Among the 1,776 base pairs, 1,674 (558 amino acids) were found in the cloned DNA fragment, and 102 base pairs (34 amino acids) were in the vector pUB110 DNA. The COOH terminal region of the alpha-amylase of pTUB4 was encoded in pUB110. The electrophoretic mobility in a 7.5% polyacrylamide gel of the alpha-amylase was slightly faster than that of the parental alpha-amylases. The NH2 termination portion of the gene encoded a 41-amino acid-long signal sequence (Ohmura et al., Biochem. Biophys. Res. Commun. 112:687-683, 1983). The DNA sequence of the mature extracellular alpha-amylase, a potential RNA polymerase recognition site and Pribnow box (TTGATAGAGTGATTGTGATAATTTAAAAT), and an AT-rich inverted repeat structure which has free energy of -8.2 kcal/mol (-34.3 kJ/mol) were identified. The AT-rich inverted repeat structure seemed to correspond to the hyperproducing character. The nucleotide sequence around the region was quite different from the promoter region of the B. subtilis 168 alpha-amylase gene which was cloned in the Escherichia coli vector systems.     

Gene for an immunoglobulin-binding protein from a group G streptococcus.

The gene (spg) for an immunoglobulin G (IgG)-binding protein from a Streptococcus clinical isolate of Lancefield group G was cloned and expressed in Escherichia coli. The complete nucleotide sequence of the gene and 5'-flanking sequences was determined. The DNA sequence includes an open reading frame which encodes a hypothetical protein of 448 amino acid residues (Mr = 47,595). The 5' end of this open reading frame encodes a sequence resembling a typical secretion signal sequence, and the remainder of the encoded protein has features reminiscent of staphylococcal protein A and of streptococcal M6 protein, including repeated sequences and a similar C-terminal structure. Aside from this C-terminal structure, the encoded protein has little direct amino acid sequence homology to either protein A or M6 protein. In E. coli, the cloned gene directs the synthesis of a protein which binds to immunoglobulins, including rabbit immunoglobulin, goat IgG, and human IgG3(lambda). Its binding properties are similar to those of the protein G described by Bj?rck and Kronvall (L. Bj?rck and G. Kronvall, J. Immunol. 133:969-974, 1984), a type III Fc receptor from a group G streptococcus.     

Nucleotide sequence of the gene for an alkaline endoglucanase from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis.

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M(r) of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M(r) of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGCGGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

Molecular cloning and characterization of an alkalophilic Bacillus sp. C125 gene homologous to Bacillus subtilis sec Y.

A 1.8 kb HindIII DNA fragment containing the secY gene of alkalophilic Bacillus sp. C125 has been cloned into plasmid pUC119 using the B. subtilis secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs. The similarity of these ORFs to the sequences of the B. subtilis proteins indicated that they were the genes for ribosomal protein L15-SecY-adenylate kinase, in that order. The gene product of the alkalophilic Bacillus sp. C125 secY homologue was composed of 431 amino acids and its M(r) value has been calculated to be 47,100. The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments. The total amino acid sequence of alkalophilic Bacillus sp. C125 secY homologue showed 69.7% homology with that of B. subtilis secY. Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmic domains (73% identity) with less homologous regions present in extracellular domains (43% identity).     

Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis.

The structural gene for a subtilisin J from Bacillus stearothermophilus NCIMB10278 was cloned in Bacillus subtilis using pZ124 as a vector, and its nucleotide sequence was determined. The nucleotide sequence revealed only one large open reading frame, composed of 1,143 base pairs and 381 amino acid residues. A Shine-Dalgarno sequence was found 8 bp upstream from the translation start site (GTG). The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprised of 275 residues. The productivity of subtilisin in the culture broth of the Bacillus subtilis was about 46-fold higher than that of the Bacillus stearothermophilus. The amino acid sequence of the extracellular alkaline protease subtilisin J is highly homologous to that of subtilisin E and it shows 69% identity with subtilisin Carlsberg, 89% with subtilisin BPN' and 70% with subtilisin DY. Some properties of the subtilisin J that had been purified from the Bacillus subtilis were examined. The subtilisin J has alkaline pH characteristics and a molecular weight of 27,500. It retains about 50% of its activity even after treatment at 60 degrees C for 30 min in the presence of 2 mM calcium chloride.